Transfer sheet for transferring biologically active substance to culture plate

ABSTRACT

The invention provides a biologically active substance transfer sheet which can be formed with simple steps, a cell culture kit which comprises a cell culture plate and a biologically active substance transfer sheet, a producing method therefor, and a method for screening cell culture conditions with cells. The transfer sheet is prepared by providing biologically active substances having biological activity in plural areas on a sheet base, and biologically active substances are supplied from the sheet to culture regions provided on the plate.

TECHNICAL FIELD

The present invention relates to a sheet for transfer one or morebiologically active substances being physiologically active to a certaincell, which sheet is usable for identifying their functions or theeffect of combinations of two or more biologically active substances orfor screening biologically active substances utilizing cells. Thepresent invention also relates to a cell culture kit constituted of atleast a cell culture plate and the above biologically active substancetransfer sheet, a producing method thereof and a method for screeningcell culture conditions utilizing the same.

BACKGROUND ART

In recent years, studies of culturing animal or plant cells undervarious conditions and studies of products of cultured cells have beenactively carried out. Particularly, studies on production of substances,of which artificial synthesis is impossible or very difficult, utilizingcertain cell activities are carried out in various fields. Also studiesare carried out to identify substances that affect cellular growth anddifferentiation so as to obtain proliferation or differentiation ofcertain cells with them according to the purpose. Also with the rapidprogress in cell engineering and medical engineering, minute biosensors,artificial organs, neurocomputers and the like are attracting attentionsand actively studied.

In order to utilize cells in vitro as explained above, it is essentialto dispose cells to control their proliferation, differentiation andsubstance production in a desired manner. However, the mechanisms ofcell disposition, cell proliferation and differentiation, and substanceproduction have not been sufficiently clarified, so that the cellculture under controlled conditions is extremely difficult impedingresearches utilizing cells.

Also tailor-made therapy considering individual difference in thesensitivity to drugs, of which concept has recently been widelyrecognized, is strongly desired, but the influence of biologicallyactive substances has been investigated on the function of respectivesubstances, particularly because of technical reasons, and there has notbeen established an effective method of easily investigatingpresence/absence of effects of plural drugs, or a required dose thereofor a combined effect thereof.

As an attempt to control cell disposition, U.S. Pat. No. 6,368,838discloses a method for culturing cells by forming a pattern of celladhesive portions on a substrate to control arrangement of cells ofplural species. U.S. Pat. No. 5,108,926 also describes formation of apattern on a substrate by applying a cell-adhering protein using an inkjet printer and cell culture thereon. These methods allow to; culturecells on the formed pattern of the cell-adhering protein, but not tocontrol the proliferation, differentiation and substance production ofthe cells or to achieve screening of cell culture conditions. Also“Protein, Nucleic acid and Enzyme”, vol. 45, 727-734(2000) describesimmobilization of a cell growth factor, which affects cell proliferationor differentiation, on a substrate using photolithography, therebyinvestigating its influence on the cell proliferation anddifferentiation. However, the substrate on which the cell growth factoris immobilized is not used for screening, and the photolithography hasproblems that a rare biological substance is wasted and the productionprocess is complicated requiring repetition of exposure and developmentsteps.

Japanese translation of PCT international application No. 2000-512009proposes a cell screening method by immobilizing onto a substrate asubstance that affects cell adhesion. In this method, reactivefunctional groups provided on the substrate and the cell-adheringsubstance are bonded through a divalent crosslinking reagent, andphotolithographic technology is used to bond the reactive functionalgroup and the cell-adhering substance. However it has a problem, inaddition to the aforementioned problems, that when plural cell-adheringsubstances are immobilized, it is extremely difficult to avoid asituation that an already immobilized substance and a substance to benewly immobilized are bonded by the divalent crosslinking reagent inundesired positions, that is, it is extremely difficult to arrangecell-adhering substances in desired positions. Also the proposed methodis not to immobilize a substance influencing the cell proliferation,differentiation and substance production. That method is to screen cellsby immobilizing cells in individual wells through the immobilizedadhering substance, culturing the cells in a culture medium anddetecting a certain substance produced by the cells. Thus it is notintended for screening a substance which influences at least one ofadhesive property, proliferation, differentiation, survival, maintenanceof an undifferentiated state, death and material production of cells, asintended in the present invention.

U.S. Pat. No. 6,103,479 discloses a method that allows effectiveconfirmation of effects of a physiologically active substance usingcells, where different cells are immobilized on a substrate to form anarray to which an physiologically active substance is applied. Thismethod, however, does not consider immobilization of physiologicallyactive substances onto a substrate. This method requires a microfluidicdelivery system to delivery the physiologically active substance to thesubstrate, and when plural physiologically active substances are used,complicated operations are required to load them onto the apparatus.

Also Japanese Patent Application Laid-open No. 2062-355025 discloses amethod of forming a screening substrate characterized in immobilizingplural screening substances by using liquid discharge means in desiredareas of a base, thereby providing different screening functions. Inthis invention, since the substances for screening are immobilized tothe screening substrate, cells often cannot intake the screeningsubstance into the cells. Therefore, this invention is not effective inthe case where only the screening substance taken into the cells canaffect at least one of the cellular functions including proliferation,differentiation, survival, maintenance of an undifferentiated state,death and substance production.

Also Japanese Patent Application Laid-open No. 2002-328124 discloses ascreening method with a higher order combination of biological activesubstances, but this is to evaluate an effect of a function ofbiological active substances provided to a substrate, so it cannot beused for screening effects of biological active substances that functionin vivo in a state immobilized on the extracellular matrix, or an effectinduced by successive addition of biological active substances.

Also Japanese Patent Application Laid-open No. 2003-33177 proposes asimple assay of chemical substances such as drugs or toxic substances,preparing a cell array divided into plural areas and providing abiologically active substance to each area to carry out simultaneousscreening of plural samples. In such a method, however, eachbiologically active substance is provided to the cultured cells by usinga dispensing means. Thus there is a risk of contamination in thedispensing step and it requires a specific apparatus for dispensing thebiologically active substances, far from convenient use.

DISCLOSURE OF INVENTION

In consideration of the foregoing, the present invention aims to providea sheet for transfer one or more biologically active substances and acell culture kit using it which can solve the technical problems in theaforementioned prior techniques and enable simultaneous evaluation ofthe effects of plural biologically active substances in an immobilizedor dissolved state through simple steps, as well as a producing methodof such a sheet and a screening method utilizing the same, therebyproviding a basic technology for further advance in cell engineering andfor various cell-utilizing devices. Another object of the presentinvention is to provide a screening method utilizing such a cell culturekit, for screening a substance which influences at least one of all thebiological activities of a cell. Still another object of the inventionis to provide a method of screening a biologically active substanceutilizing cells.

Still another object of the present invention is to provide a methoduseful in screening of a biologically active substance which influencescell functions, at least one of proliferation, differentiation,survival, maintenance of an undifferentiated state, death and substanceproduction, only when it is taken into cells; or a method useful indetermining effects of successive addition of one or more biologicallyactive substances.

The present invention provides a biologically active substance transfersheet (hereinafter referred to as a transfer sheet) including thefollowing embodiments:

-   (1) A transfer sheet that transfers a biologically active substance    to a culture region on a culture plate when placed on the culture    plate, the sheet which comprises a sheet base; and a holding area    provided on the sheet base, the area holding at least one substance    having a biological activity to a cell; wherein the holding area is    provided in a position for covering the culture region of the    culture plate.-   (2) The transfer sheet of the above (1), wherein the biologically    active substance is releasable from the sheet.-   (3) The transfer sheet of the above (1), wherein the sheet contains    two or more holding areas.-   (4) The transfer sheet of the above (3) wherein the holding areas    hold different biologically active substances or different    combinations of two or more biologically active substances.-   (5) The transfer sheet according to the above (3), wherein the    holding areas hold a biologically active substance in different    concentrations.-   (6) The transfer sheet according to the above (1), wherein the sheet    base is made from an elastic or flexible film at least at the    holding area.-   (7) The transfer sheet according to the above (1), wherein the    holding area is a protruding portion provided on the sheet base, and    the biologically active substance is held on the protruding area.-   (8) The transfer sheet according to the above (1), wherein a holding    layer is formed on an entire or partial surface of the sheet base    for holding a biologically active substance thereon.-   (9) The transfer sheet according to the above (1), wherein the    holding area is able to release the biologically active substance in    a sustainable manner or the area releases a biologically active    substance provided with a property for sustained release.-   (10) The transfer sheet according to (1), wherein each holding area    or a group of two or more areas is surrounded by a protruding wall    structure.-   (11) A method for producing a transfer sheet according to any of (1)    to (10), the method comprising a step of providing a holding area    with a biologically active substance by using liquid discharge    means.-   (12) The method according to (11), wherein the liquid discharge    means is discharge means by a thermal ink jet method.-   (13) The method according to (11), wherein the liquid discharge    means is discharge means by a piezo ink jet method.-   (14) The method according to any of (11)-(13), further comprising a    step of immobilizing the biologically active substance by applying    an immobilizing energy from the exterior.-   (15) A method for screening cell culture conditions utilizing a    transfer sheet of any of (1) to (9), the method comprising the steps    of placing the transfer sheet on a plate having at least one culture    region to cover the culture region containing a culture liquid with    a holding area holding a biologically active substance on the    transfer sheet; and supplying the culture liquid with the    biologically active substance from the holding area.-   (16) The screening method according to (15), further comprising a    step of replenishing the culture liquid with a substance necessary    for screening.-   (17) The screening method according to (15) or (16), further    comprising a step of replacing the sheet with another transfer sheet    of a same or different type.-   (18) The screening method according to any of (15)-(17), further    comprising a step of observing a morphological change of the cell.-   (19) The screening method according to (18), wherein cells are    stained for evaluation.-   (20) The screening method according to any of (15)-(19), further    comprising a step of executing a quantitative determination of a    substance synthesized in the cell.-   (21) The screening method according to any of (15)-(19), further    comprising a step of executing a quantitative determination of a    substance incorporated in the cell.-   (22) The screening method according to (20) or (21), wherein the    step of executing a quantitative determination is carried out by at    least one of a radiation intensity measurement, a fluorescence    intensity measurement, a luminescence intensity measurement and an    optical absorbance measurement.

The present invention also provides a cell culture kit including thefollowing embodiments. Such a cell culture kit comprises a culture platehaving at least one culture region for culturing a cell; and a sheethaving a portion to cover the culture region; wherein each of theculture region and the covering portion holds at least one substancehaving a biological activity to a cell, and at least one biologicallyactive substance held by the culture region or the covering portion isimmobilized thereon.

The kit may contain two or more combinations of the culture regions andthe covering portions.

The cell culture kit may have a constitution wherein the biologicallyactive substance held in the culture region is immobilized thereto, thebiologically active substance held in the covering portion is attachedso as to be released in contact with a culture liquid, and the sheetbase and the covering portion constitute a transfer sheet fortransferring the biologically active substance to the culture region.

The present invention also provides a method for producing a cellculture kit of the aforementioned configuration characterized in that atleast liquid discharge means is utilized for providing the cultureregion and the covering portion with the biologically active substance.The liquid discharge means is of a thermal ink jet method, or of a piezoink jet method.

The present invention also provides a screening method utilizing a cellculture kit of the aforementioned configuration, where the methodcomprises the steps of placing a sheet having a covering portion on aplate having a culture region to cover the culture region with thecovering portion, culturing a cell in the culture region covered withthe covering portion in contact with a first biologically activesubstance immobilized one of the culture region and the coveringportion; and supplying the culture liquid with a second biologicallyactive substance attached to the rest of the culture region and coveringportion. The cell culture kit of the invention allows secure dispositionof a biologically active substance to a desired position by a simpleprocess, and can be utilized for screening (evaluating) the effects ofvarious biologically active substances for the cells.

More specifically, screening with the cell culture kit of the inventionallows to specify a factor necessary for proliferation, differentiation,survival, maintenance of undifferentiated state, death or substanceproduction, whereby an efficient cell culture method can be determined.Also according to the present invention, substances can be screened notonly in their solid phase but also in their liquid phase, that is,screening is carried out under conditions closer to in vivo conditionsusing combinations of biologically active substances in theirimmobilized state or dissolved state. The present invention also allowsinvestigation of difference in the effect of a substance in its solidphase and in its liquid phase or combination thereof, and evaluation ofindividual sensitivity to a drug or an endocrine perturbing substance,so-called environmental hormone. It is also possible, based on theresult of such evaluation, to determine a tailor-made therapeutic methodfor individuals. It is furthermore possible to screen useful substanceshaving biological activities to cells using cells.

The transfer sheet and the cell culture substrate of the invention canbe produced employing an ink jet method as the liquid discharge means,enabling simultaneous action of plural substances on the cells atvarious concentrations by choosing liquid species and controlling anumber of liquid droplets. Also the transfer sheet of the inventionallows precise transfer of plural substances in a cell culture liquid,enabling exact evaluation of an effect of a system comprised of pluralbiologically active substances to the cell proliferation,differentiation and survival, which has been difficult to evaluate inthe prior technology. Also a successive addition of biologically activesubstances or change in combination is easy. Therefore, the effect ofsuch conditions can be studied easily and effectively. Furthermore, thecell culture substrate of the invention can be easily produced in alarge scale, and stored stably and used whenever desired as a screeningsubstrate.

Other features and advantages of the present invention will be apparentfrom the following description taken in conjunction with theaccompanying drawings, in which like reference characters designate thesame or similar parts throughout the figures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the description, illustrate embodiments of the invention and,together with the description, serve to explain the principles of theinvention.

FIGS. 1A and 1B illustrate an example of a producing method of abiologically active substance transfer sheet of the present invention;

FIG. 2 illustrates an example of biologically active substance formingareas in a biologically active substance transfer sheet;

FIG. 3 illustrates an example of attaching a biologically activesubstance transfer sheet to a 96-well transparent microplate to transferthe biologically active substance to the culture medium;

FIG. 4 is a cross-sectional view of a state where a biologically activesubstance transfer sheet is attached to a 96-well transparentmicroplate;

FIG. 5 illustrates an example of transfer of a biologically activesubstance from a biologically active substance transfer sheet to atransparent microplate;

FIGS. 6A, 6B and 6C illustrate an example of a method for producing acell culture kit by using liquid discharge means; and

FIGS. 7A and 7B illustrate an example of a cell culture processutilizing a cell culture kit.

BEST MODE FOR CARRYING OUT THE INVENTION

Preferred embodiments of the present invention will now be described indetail in accordance with the accompanying drawings.

First, there will be explained an example of a biologically activesubstance transfer sheet (transfer sheet) of the invention. As shown inFIG. 1A, the transfer sheet 1 is constituted of a base (substrate) 11and two or more biologically active substances 12 which are positionedin desired positions of the base 11 with different densities orconcentrations forming a pattern. FIG. 2 shows a transfer sheet 1 onwhich two or more biologically active substances 12 are patterned in 96areas (101-196). Thus, when two or more active substances 12 provided inone area of the pattern come into contact with the culture liquid in aregion corresponding that area, the active substances are liberated anddissolved in the culture liquid, in which the cells are cultured.Accordingly, after the culture, one can observe difference in at leastone of cell adhesion, proliferation, differentiation, survival,maintenance of the undifferentiated state, death, and substanceproduction induced by various combinations of the biologically activesubstances 12. One of the advantages obtained by preparing the transfersheet with the liquid discharge means is that the biologically activesubstances can be easily positioned in a patterned area with anarbitrary ratio.

A cell employable in the present invention may be any procaryotic oreucaryotic cells. For example, there can be employed a bacteria cell, anyeast cell, a protozoa cell, a neuron, a fibroblast, a smooth musclecell, a skeletal muscle cell, a gliocyte, an embryonic stem cell, ahematopoietic stem cell, a mast cell, a fat cell, a nerve stem cell oran immune cell including T cell and B cell, or a cluster thereofincluding transformed or non-transformed cells.

The biologically active substance 12 is a substance that affectscellular functions such as adhesion property, proliferation,differentiation, survival, maintenance of undifferentiated state, death(apoptosis), substance production, gene expression and cellular signaltransmission. Examples of such substance include an extracellular matrixprotein, an antibody having a specific binding ability to the cellsurface, a cytokine, and a chemical substance that affects cellproliferation or differentiation when taken into the cells. Examples ofthe extracellular matrix protein include collagen, fibronectin andlaminin. Cytokine includes a cell growth factor and a hormone, and thecell growth factor includes a nerve growth factor (NGF), an epitheliumgrowth factor (EGF), a basic fibroblast growth factor (bFGF), a bonemorphogenetic protein (BMP-2), an insulin-like growth factor (IGF-I),and a tumor necrosis factor (TNF). Also examples of hormone include apeptide such as insulin or calcitonin, a steroid such as aldosterone orprogesterone, and an amino acid derivative such as epinephrine orthyroxine. There are also included other chemical substances such asallergen which induces allergy, and so-called endocrine-perturbingsubstances. There are further included antibiotics which can suppressbacterial growth. There are further included an antibody thatspecifically binds to the aforementioned substance thereby allowing toinvestigate localization of such a substance in the cell, and aneutralizing antibody capable of suppressing the function of suchsubstance. On the base 11, various combinations of plural differentbiologically active substances can be provided in different areas. Onthe base 11, there may be formed, not only areas which are different inthe combination of plural different biologically active substances, butalso areas in which the density of the biologically active substance 12differs.

When such areas come into contact with a culture liquid in the regionscorresponding to the areas, the active substance 12 is liberated anddissolved in the culture liquid, in which cells are cultured. FIG. 3shows an example. The transfer sheet 1 having the pattern of theaforementioned 96 areas covers a microplate 2 (96-well) containing acell culture liquid in each well, in such a manner that each of the 96areas of the transfer sheet 1 covers the corresponding well 21. FIG. 4is a schematic cross-sectional view of a mutually adhered state.

Then the sheet 1 and the microplate 2 in a superposed state are vibratedor rotated to liberate and dissolve the active substance held on thesheet 1, into the cell culture liquid. Then, after sheet 1 is removedfrom the microplate, a cell suspension is added to each well to culturethe cells. After the culture, one can observe in detail any differencein cell adhesion, proliferation, differentiation, survival, maintenanceof an undifferentiated state, cell death, or substance productionaccording to combinations of the active substances 12 or densitiesthereof. In the above example, the contact was carried out immediatelybefore the start of culture. However, in the invention, the transfersheet may be brought into contact with the culture liquid 4 at anarbitrary timing after the start of the cell culture as shown in FIG. 4,to liberate and dissolve the active substance into the culture liquid.

The base 11 can be made of any material of any shape, as long as it canhold the active substance. More specifically, a glass plate, a plasticplate, a plastic sheet, a polymer film or a paper can be employedadvantageously. Also the base 11 may be transparent, opaque, or colored.Also the base 11 may be formed by a stretchable, elastic or flexiblefilm. In such a case, synthetic rubber such as silicone rubber, naturalrubber or a paraffin film can be advantageously employed. When the areasholding the active substance on the transfer sheet are brought intoclose contact with the cell culture wells for achieving the contact withthe culture liquid therein and for liberating and dissolving the activesubstance therein, a stretchable or elastic sheet is preferable as itprovides satisfactory adhesion to prevent leakage of the liquid. Also itis possible to press down the stretching or flexible transfer sheet,using a pointed article from the above until the sheet comes in contactwith the surface of the culture liquid 4, thereby dissolving the activesubstance into the culture liquid as shown in FIG. 5. In this case,there is obtained an advantage that the biologically active substancecan be liberated and dissolved without rotation or shaking.

Also in the base 11, each area in which the active substance 12 ispositioned, or a group of two or more areas, may be in a recess. Such aconfiguration facilitates positioning of liquid droplets by liquiddischarge means. A base having such recess can by formed by a molding ofa resinous material or an etching method utilizing a photolithographictechnology.

Also in the base 11, each area in which a biologically active substance12 is positioned, or a group of two or more areas, may be surrounded bya wall structure. Such configuration can facilitate positioning ofliquid droplets by liquid discharge means. A base having suchwall-shaped structure can by formed for example by photolithography.Alternatively, the base 11 may have protruding portions, and the activesubstance 12 may be provided on such protruding portions. In such acase, the active substance 12 can be easily dissolved in the cultureliquid 4 by merely inserting the protruding portion into a culture well.

Such protruding shape can be prepared by press molding with a metalmold. Also an adhesive material, or a water repellent material may beapplied or printed in an area where the active substance is notpositioned. Also, as shown in FIG. 1B, a holding layer 14 which holdsthe biologically active substance may be formed on an entire or partialsurface of the base 11.

The holding layer 14 is to help prompt release and dissolution of thebiologically active substance on contact with the culture liquid in theculture well. Therefore the holding layer 14 is preferably made of awater-soluble or water-swelling substance, which preferably can hold thebiologically active substance stably. Preferable examples include asynthetic polymer such as polyvinyl alcohol, polyethylene glycol, apolyacrylic acid salt, a polymethacrylic acid salt, a methacrylic acidcopolymer, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose phthalate,cellulose acetate phthalate, carmerose, or polyvinylpyrrolidone, astarch derivative such as starch, hydroxyethyl cellulose, sodiumalginate, sodium celluloseglycollate, or sodium starchglycollate, anatural polymer such as dextrin, gum Arabic, carrageenan, agar, gelatin,tragacanth or crystalline cellulose, or a water-soluble compound such asglucose, sucrose, fructose, or xylitol. Such a material may be appliedon an entire or partial surface of the base 11 as required. The holdinglayer 14 can be coated, for example, by using bar coating, bladecoating, screen printing, flexographic printing or offset printing. Thethickness of the holding layer can be arbitrarily selected, but ispreferably about 1 to 100 μm.

By suitably selecting the configuration of the holding layer, it ispossible to attain sustained release of the biologically activesubstance from the holding layer into a culture liquid. Such sustainedor controlled release may also be achieved by adding, a substanceenabling controlled release such as a water-soluble styrene-acrylicresin to a liquid containing the biologically active substance, whichliquid is used at application of the biologically active substance tothe sheet.

Such a transfer sheet 1 can be prepared in the following manner (cf.FIGS. 1A and 1B). First, a base 11 may be subjected to a pretreatmentmentioned above if necessary. More specifically, the base 11 is washedto eliminate undesired materials and may be subjected to variouschemical or physical treatments such as UV irradiation or coronadischarge.

The biologically active substance 12 is deposited either directly onsuch base 11, or on a sheet prepared by forming a holding layer 14 on anentire or partial surface of the base 11. Liquid discharge means 13 isemployed for depositing. The liquid discharge means 13 is capable ofdischarging a liquid droplet of a volume of 100 nl or less per drop,more specifically 1 nl or less, such as a micropipette, amicrodispenser, or a discharge apparatus of ink jet method. A dischargeapparatus of ink jet method can be employed particularly advantageouslybecause the discharge apparatus is available inexpensively and a minuteliquid droplet can be discharged. Furthermore, among the ink jetmethods, a thermal ink jet method and a piezo ink jet method can beemployed advantageously. A discharge apparatus of the thermal ink jetmethod, being easy in preparation of fine discharge ports, can dischargea liquid containing a biologically active substance 12 in predeterminedpositions at a high density. Also a discharge apparatus of the piezo inkjet method, in which a discharge energy is generated by a displacementof a piezoelectric element, can discharge the biologically activesubstance 12 without giving a thermal stress thereto.

The biologically active substance 12 is dissolved or dispersed in anappropriate solvent for discharge. Any solvent (dispersion medium) maybe employed as long as it can stably dissolve or disperse thebiologically active substance 12, but water is employed advantageously.Water content is 30 mass % or higher, preferably 50 mass % or higher.Preferably, water is ion-exchanged water (deionized water) or variousbuffers for stably dissolving the biologically active substance 12.Also, if necessary, a water-soluble solvent may be employed. The amountof each water-soluble solvent to be added is 50 mass % or less,preferably 30 mass % or less. Any water-soluble solvent may be employedas long as it is soluble in water, and examples include an alkyl alcoholwith 1 to 4 carbon atoms such as methyl alcohol, ethyl alcohol, n-propylalcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, ort-butyl alcohol; an amide such as dimethylformamide ordimethylacetamide; a ketone or a ketoalcohol such as acetone ordiacetone alcohol; an ether such as tetrahydrofuran or dioxane; anpolyalkylene glycol such as polyethylene glycol, or polypropyleneglycol; an alkylene glycol in which an alkylene group has 2-6 carbonatoms such as ethylene glycol, propylene glycol, butylenes glycol,triethylene glycol, 1,2,6-hexanetriol, thiodiglycol, hexylene glycol ordiethylene glycol; glycerin; a lower alkyl ether of a polyhydric alcoholsuch as ethylene glycol monomethyl ether, ethylene glycol monoethylether, ethylene glycol monobutyl ether, diethylene glycol monomethylether, diethylene glycol monoethyl ether, diethylene glycol monobutylether, triethylene glycol monomethyl ether, triethylene glycol monoethylether, or triethylene glycol monobutyl ether; N-methyl-2-pyrrolidone,2-pyrrolidone and 1,3-dimethyl-2-imidazoline. These solvents may besuitably selected in one or more kinds. Among these water-solubleorganic solvents, there is preferred a polyhydric alcohol such asdiethylene glycol, or a lower alkyl ether such as triethylene glycolmonomethyl ether. In case of thermal ink jet method, addition of analcohol such as ethanol or isopropyl alcohol or a lower alkyl ether of apolyhydric alcohol is advantageous for more stable bubble formation on athin film resistor in a discharge port of the ink jet head, forproviding the biologically active substance.

The solution of the biologically active substance 12 may furtherinclude, if necessary for obtaining desired physical properties, asurfactant, an anti-foaming agent, an antiseptic, an inorganic salt, anorganic salt and the like.

As to the surfactant, any surfactant not detrimentally influencing thebiologically active substance 12 for example on a storage stability maybe employed, for example an anionic surfactant such as a fatty acidsalt, a higher alcohol sulfate ester salt, a liquid fatty oil sulfonateester salt, or an alkylallylsulfonic acid salt, or a nonionic surfactantsuch as an polyoxyethylene alkyl ether, a polyoxyethylene alkyl ester, apolyoxyethyelensorbitan alkyl ester, acetylene alcohol or acetyleneglycol, and these may be employed singly or in a mixture of two or morekinds.

In order to release the biologically active substance 12 gradually fromthe transfer sheet, a hydrophilic polymer compound may be added to thesolution in addition to the foregoing components. For example, polyvinylalcohol, a polyacrylic acid salt, a polymethacrylic acid salt, amethacrylic acid polymer, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxypropylmethyl cellulose,hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate, orpolyvinylpyrrolidone is advantageously employed for providing thecontrolled release property. Such hydrophilic resin is preferably addedwithin a range of 1 to 10 mass %, more preferably 2 to 5 mass %.

After the biologically active substance 12 is provided in a desiredposition on the base 11 by the liquid discharge means 13, the sheet maybe dried, for example, in an oven.

The transfer sheet can be prepared as explained above.

The transfer sheet can be stored in a container over a prolonged period.A more preferable stable storage can be achieved by sealing the transfersheet in a container together with a drying agent or an oxygen removingagent.

In the following, there will be explained a screening process utilizingthe aforementioned transfer sheet.

The process comprises the following steps of: contacting each areacarrying the biologically active substance of the transfer sheet withthe culture liquid in a corresponding culture well thereby transferringthe biologically active substance to the culture liquid; and culturingcells therein.

This method enables an easy transfer of the biologically activesubstance at the same time to plural culture regions (wells) of a cellculture vessel or plate. It is also possible to transfer thebiologically active substance(s) plural times during the culture periodby contacting plural transfer sheets of the same or different kind tothe culture liquid. The culture liquid may be changed when the transfersheet is replaced to remove the previously added biologically activesubstance. In such a case, it is possible to supply the samebiologically active substance to the same culture region by using atransfer sheet of the same biologically active substance, or to supply adifferent biologically active substance to change the culture conditionsby using a transfer sheet carrying a different biologically activematerial. Such a change may be carried out at any number of timesaccording to the necessity. Thus it is possible to investigate theinfluence of one or more biologically active substances to the cell indetail and in a time-series manner. Also use of the transfer sheet ofthe invention is simple and avoids contamination with undesiredsubstances.

As the culture vessel, a commercially available microplate can beadvantageously employed. The microplate contains an array of a pluralityof culture regions called wells. The number of wells can be about 12 to2,000, and can be suitably selected according to the number of thebiologically active substances to be screened and conditions thereof.For example a commercial 96-well microplate can be advantageouslyemployed in case of screening up to 96 conditions by using a cellculture.

In the transfer sheet of the invention, areas carrying the biologicallyactive substance are patterned corresponding to the positions of wellsof the microplate in such a manner that each area comes just above thecorresponding well when the sheet is superposed on the microplate. Asshown in FIG. 2, the transfer sheet is placed so as to cover themicroplate, and the biologically active substance is dissolved/extractedwith the liquid put in the well in advance. In this manner, thebiologically active substance is transferred from the transfer sheetinto each well of the microplate.

Cells are cultured in such a culture vessel under the influence of thebiologically active substance(s) on cellular functions such as celladhesion, proliferation, differentiation, survival, maintenance of anundifferentiated state, death of cells, or substance production. Thecells to be used are not particularly restricted, and any cells may beemployed for this purpose. Also one kind or two or more kinds of cellscan be used at a time for screening. Prior to the cell culture, thetransfer sheet and the cell culture vessel may be sterilized ifnecessary for example by ultraviolet irradiation or by washing withalcohol. Such an operation may prevent contamination of the culture, forexample, with undesired microorganisms.

Alternatively, a desired substance may be added to the culture liquid ofa desired region of the culture vessel 2 during the cell culture orafter the cell culture for a predetermined period, by using the transfersheet. In this manner it is possible to change proliferation,differentiation, survival, maintenance of an undifferentiated state,death, or substance production of the cells or change an adhesionproperty thereof to the substrate. Also after the cell culture, adesired substance such as an indicator may be added in a desired area.In this manner the screening can be carried out easily.

Also the cultured cells may be removed from the culture vessel duringthe cell culture therein or after cultured for a predetermined period.In this manner the removed cultured cells may be utilized as anartificially prepared tissue or a part thereof. More specifically, thecultured cells can be removed by treating the cell culture vessel withtrypsin after the cell culture. Also by coating the cell culture vesselin advance with a polymer of which hydrophilicity changes withtemperature, such as poly(N-isopropylacrylamide), and executing the cellculture thereon, it is possible to remove the cultured cells by bringingthe temperature to about 30° C., thereby changing the hydrophilicity ofthe polymer surface. In this manner the cells can be utilized forexample for a live tissue.

Next, screening means for cells cultured in the aforementioned cellculturing vessel is explained.

As screening means, morphological changes of the cells cultured in thecell culture vessel 2 can be observed. For this purpose, any microscopecapable of observing the shape of the cells can be used such as anoptical microscope a scanning electron microscope, a transmissionelectron microscope, a scanning probe microscope and a fluorescencemicroscope. A cell culture plate bearing cultured cells is placed undersuch a microscope to observe the morphology of the cells. Screening orevaluation can be simply carried out by mere microscopic observation ofthe cells. Also the cells may be stained for evaluation. Cell stainingfacilitates evaluation under a microscope when cells have proliferatedto a high density or fused by differentiation forming polykaryocytes.

In addition to the morphological observation, one can quantitativelydetermine a substance produced or incorporated by the cells while thecells adhered to the culture vessel wall and proliferated ordifferentiated thereon. In case an object substance cannot be directlydetermined, the quantitative determination may be carried out with analternative substance. For example, an object protein can bequantitatively determined by introducing a gene of a measurable proteinin the vicinity of the gene of the object protein by the geneticengineering, and by quantitatively determining the measurable protein.Thus cellular changes induced by the substance immobilized on the baseof the culture vessel can be studied in detail by measuring such aproduct, which leads to clarification of the information transmissionmechanism in the cell. When a substance incorporated into the cell isevaluated, the substance to be taken into the cells can be labeled inadvance, which makes the quantitative determination rather easy.

For a quantitative determination of these substances, there may beemployed a method of measuring radiation from a radioactive compound, amethod of measuring an amount of fluorescence emitted from a substancelabeled with a fluorescent substance, a method of measuring an amount oflight emitted from a light-emitting substance, or a method of measuringan optical absorbance of a dye.

A method that employs a radioactive compound containing a radioactiveisotope of an element abundantly present in a live tissue such ashydrogen, carbon, nitrogen, phosphor or sulfur and measures theintensity of radiation from such a compound is highly sensitive, andallows observation of phenomena occurring in a live body, because such ahot compound has the same chemical properties as the cold compound.

Also a method of labeling with a fluorescent substance is relativelysimple and gives little influence on the metabolism of the cell byemploying a fluorescent substance of a low molecular weight. Also in aquantitative determination of a substance produced by the cells by adetermination method utilizing an antigen-antibody reaction, anevaluation by a fluorescent measurement is effective since variousantibodies labeled with a fluorescent substance are available andprovide a high measuring sensitivity.

Also the method of measuring luminescence from a luminescent substanceallows to recognize even a small change, since the luminescence can bemeasured with a high sensitivity. When a gene expressed with celladhesion, proliferation, differentiation or substance production causedby a substance has been specified, it is possible to introduce a firebugluciferase gene or the like in the vicinity of such a gene and an amountof luciferase produced by the gene expression is measured from theintensity of luminescence generated on addition of ATP and luciferin. Inthis manner it is possible to evaluate the influence of the screenedsubstances from the luminescence intensity.

In a method of measuring the optical absorbance of a dye, it is possibleto amplify the optical absorbance of a dye for example by employing anenzyme reaction in combination, thereby enabling a quantitativedetermination of a substance of a very small amount.

(Embodiment of Cell Culture Kit)

A cell culture kit of the present embodiment is constituted at least ofa cell culture plate having plural divided culture regions (sections),and a sheet having covering portions for covering the culture regions.In either of the culture region and the corresponding covering portion,at least one biologically active substance having a biological activityon a cell is immobilized, and, on the rest, at least one biologicallyactive substance is held in a manner releasable to a culture liquid.Thus, the biologically active substance may be immobilized on theculture plate or to the sheet. When the active substance is immobilizedon the plate, another biologically active substance on the sheet is heldto be releasable to the culture liquid. When the active substance isimmobilized on the sheet, another biologically active substance on theculture plate is held in a releasable state to the culture liquid.

In the following, the present invention will be explained by aconfiguration in which the biologically active substance on the sheet isheld releasably to the culture liquid to constitute a transfer sheet,but the present invention is not limited thereto. A biologically activesubstance may be immobilized on the he sheet.

When a biologically active substance is immobilized to the cultureplate, the kit is constituted of a cell culture plate on which one ormore biologically active substances are immobilized in desired areas,and a transfer sheet on which one or more biologically active substancesare arranged in plural areas.

In the cell culture plate, a combination of biologically activesubstances immobilized in one region may be different between theregions. Also a density of a biologically active substance immobilizedmay be different between the regions of the cell culture plate.Furthermore, a region of the cell culture plate may immobilize pluralbiologically active substances, thereby constituting plural regions ofdifferent screening functions. Also a group of two or more regions maybe provided in a recess, or a group of two or more regions may besurrounded by a protruding wall-shaped structure.

In the transfer sheet, it is preferable that plural biologically activesubstances are releasable from the sheet. The transfer sheet maycontain, in the plural areas thereof, plural areas different in thecombination of the biologically active substances. The transfer sheetmay contain, in the plural areas thereof, plural areas different in adensity of the biologically active substance. The sheet base may beconstituted of a stretchable or flexible sheet at least in an areaholding the biologically active substance. In the transfer sheet, aholding layer for carrying the biologically active substance may beformed on an entire or partial surface of the sheet base. Also in thetransfer sheet, each area may be formed in a recess. Also in thetransfer sheet, each area may be formed on a protruding portion formedon the sheet. Also in the transfer sheet, each area or a group of two ormore areas may be surrounded by a protruding wall-shaped structure.

The cell culture plate and the transfer sheet can be prepared by amethod including a step of positioning each biologically activesubstance by liquid discharge means in plural regions of the cellculture plate or in plural areas in each region, and/or in plural areason the releasable transfer sheet. To produce the cell culture plate andthe transfer sheet, the thermal ink jet method or the piezo ink jetmethod can be employed as the liquid droplet discharge means. Also anenergy for immobilizing the biologically active substance to the cellculture plate may be applied from the exterior.

A screening method utilizing the transfer sheet includes steps ofculturing cells in a culture liquid in contact with a regionimmobilizing a biologically active substance of the cell culture plate,contacting the culture liquid with an area holding a biologically activesubstance of the transfer sheet thereby releasing and dissolving thebiologically active substance into the culture liquid in which the cellsare cultured. The screening method may include a step of replenishing asubstance necessary for the screening, by utilizing plural transfersheets. In such a case, it is possible to supply the same biologicallyactive substance to the same culture region by replacing with the sametransfer sheet during the culture, or to change the culture conditionsby changing the sheet to a different sheet to add a differentbiologically active substance. Such change may be any number of timesaccording to the necessity. Also there may be included a step ofexecuting a culture under a gradual supply of the biologically activesubstance to the cells in the culture liquid, by employing a transfersheet in which a biologically active substance is provided on a holdinglayer that enables sustained release and contacting such a sheet withthe culture liquid during an entire culture period or a part thereof.

Screening in the screening method of the present embodiment can becarried out, for example, based on at least one of following items:

-   -   (1) A change in cell morphology is evaluated in a desired area        of the plate.    -   (2) A substance incorporated in the cells is quantitatively        determined in a desired area of the plate.    -   (3) A substance synthesized in the cells is quantitatively        determined in a desired area of the plate.    -   (4) A shape evaluation or a quantitative determination of a        substance is carried out by cell staining.    -   (5) A signal by a reporter gene is detected.    -   (6) An evaluation is carried out by a site blot assay.    -   (7) A quantitative determination of a substance is carried out        by a measurement of a radiation intensity.    -   (8) A quantitative determination of a substance is carried out        by a measurement of a fluorescent intensity.    -   (9) A quantitative determination of a substance is carried out        by a measurement of a luminescent intensity.    -   (10) A quantitative determination of a substance is carried out        by a measurement of an optical absorbance.

The embodiments will be explained in the following in more detail.

In the following there will be explained an example of the cell culturekit of the present invention. As shown in FIG. 6A, in a cell cultureplate 2, one or more biologically active substances 22 (3 kinds in FIG.6A) are provided in desired regions formed on a base (substrate) 20, andeach biologically active substance 22 is immobilized on the base 20. Onthe other hand, in a transfer sheet 1, two or more biologically activesubstances 12 are provided on a sheet shown in FIG. 6B, at desiredpositions corresponding to the regions of the cell culture plate. It isalso possible, as shown in FIG. 6C, to provide the biologically activesubstance 12 on a protruding portion 23 formed on the sheet. Forpositioning, liquid droplet discharge means 13 can be utilized. Inculturing the cells with the cell culture plate 2, prior to the start ofthe culture or at an arbitrary timing after the start of the culture,the transfer sheet is placed so as to cover the cell culture plate 2 asshown in FIG. 7A, and the sheet is depressed from the above to achieve acontact with the culture liquid 4 and the sheet, thereby releasing anddissolving the biologically active substance in the culture liquid,whereby the biologically active substance 22 immobilized on the cellculture plate and the biologically active substance 12 provided on thetransfer sheet act on the cells and there can be inspected ahigher-order effect of a combination of the biologically activesubstances (12, 22) in the immobilized and dissolved states. Also incase of employing a sheet in which the biologically active substance 12is provided on the protruding portion 23 formed on the sheet (FIG. 6C),it is possible to contact the area of the biologically active substancewith the culture liquid 4 to induce liberation and dissolution by merelyplacing the sheet 1 to cover the cell culture plate 2 (FIG. 7B).

A cell employable in the present embodiment may be any procaryotic oreucaryotic cell, and such cells may be suitably selected.

In the present embodiment, the biologically active substances 22, 12 canbe the aforementioned substances for culture control, and such substancemay be arbitrarily immobilized to the base 20 or positioned on the sheet10 in consideration of the action to the cells or localization in thecells.

Also as to the biologically active substance 22 and/or the biologicallyactive substance 12, a combination of the biologically active substances22 and 12 may be different depending on an area/region or on a group oftwo or more areas/regions of the base 20 and the sheet 10. It is thuspossible to evaluate at least a difference in the effects in thecombination of the biologically active substances 22 and 12 to thecells.

Also the biologically active substances 22 and 12 may be different indensity according to the position on the base 20 and the sheet 10. Inthis manner it is possible to evaluate in more detail difference of thebiological activity on the cells due to the density of the biologicallyactive substance 22 and/or a difference in the concentration of thebiologically active substance 12 in the culture liquid.

One of the advantages obtained by the use of the liquid discharge meansis that the biologically active substance can be easily positioned in anarea with an arbitrary amount.

The immobilization of the biologically active substance 22 on the base20 may be achieved by a covalence bond, an electrostatic attractiveforce or a biological affinity. Immobilization by a covalence bond tothe base 20 allows strong immobilization of the biologically activesubstance 22, of which bonding is scarcely affected by the cells or bythe culture liquid, whereby a stable immobilization on the base 20 canbe attained.

The base 20 and the sheet 10 can be made of any material of any shape,as long as the biologically active substance can be stably immobilizedor held. More specifically, a glass plate, a plastic plate, a plasticsheet, a polymer film or a paper can be employed advantageously. Alsothe base 20 and the sheet 10 may be transparent, opaque, or colored.Also for immobilizing the biologically active substance 22 on the base20 or for improving stability of the biologically active substances 22,12 on the base 20 and the sheet 10, a treatment with a chemical materialor irradiation may be applied on the entire surface or a part thereof.Also an adhesive material, or a water repellent material may be appliedor printed in an area where the biologically active substance is notprovided.

The sheet 10 may be formed with a stretchable film. For example,synthetic rubber such as silicone rubber, natural rubber, latex, apolyolefin film such as of polyethylene, polymethylpentene or aparaffinic film can be advantageously employed. The biologically activesubstance can be liberated and dissolved in the culture liquid bycontacting the wall-shaped structure, formed on the cell culture plate,so as to surround the area on the sheet 10 where the biologically activesubstance is provided, and depressing the sheet from the rear surfaceuntil such area comes into contact with the culture liquid. In suchcase, a stretchable or elastic sheet is preferable as it provides asatisfactory adhesion thereby avoiding the leakage of the liquid.

A chemical treatment or an electrostatic treatment may be applied to thebiologically active substance or to the sheet, in order that thebiologically active substance 12 on the sheet 10 can be easily liberatedfrom the sheet. For transferring the biologically active substance fromthe sheet to the culture liquid, after the sheet is adhered to the wallstructure on the cell culture plate 2 and the cell culture plate and thesheet are shaken in such a manner that the culture liquid comes intocontact with the area where the biologically active substance isprovided, or the sheet is pressed with a rod-shaped article from theabove thereby contacting the area holding the biologically activesubstance with the culture liquid (FIG. 7A). Also for efficientliberation, a vibration may be applied to the culture liquid or to thesheet. Also the transfer sheet may have a holding layer, for carryingthe biologically active substance, on the sheet base. In this manner, itis possible to stably hold the biologically active substance. Also thesheet may have a recess for carrying the biologically active substance,which may be provided in such a recess. Also the sheet may be providedwith a protruding wall-shaped structure, which surrounds each area or anarea group constituting of two or more areas. Such configuration canfacilitate positioning of a liquid droplet, provided by liquid dischargemeans. Furthermore, the sheet may be provided with a protruding portion,and the biologically active substance may be provided thereon. In such acase the biologically active substance can be easily contacted with theculture liquid and can be transferred thereto.

Formation of a recess, a protruding wall-shaped structure, or aprotruding portion on the transfer sheet can be achieved for example byan injection molding, a pour molding, an adhesion of a chip by thermalfusion or with an adhesive material, or a press molding with a metalmold.

The biologically active substance 22 immobilized on the cell cultureplate 2 and the biologically active substance 12 provided on thetransfer sheet 1 may be constituted of the same biologically activesubstances, or different biologically active substances, or some of thebiologically active substances are the same.

The cell culture plate 2 and the transfer sheet 1, described above, canbe prepared in the following manner. First, a base 20 and a sheet 10 maybe subjected to the treatment mentioned above if necessary. Morespecifically, the base 20 and the sheet 10 are washed to eliminateundesired substances and may be subjected to various chemical orphysical treatments such as UV irradiation or a corona discharge. Alsofor the cell culture plate, it is possible, if necessary, to apply apolymer material or a silane coupling agent on the base 20 or a partthereof.

A treatment for facilitating carrying of the biologically activesubstance may be applied to the sheet 10. For such a treatment, abiologically active substance-holding layer may be formed on the entiresurface of the sheet. For this purpose, there can be advantageouslyemployed, for example, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, polyvinylpyrrolidone,macrogol, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, amethacrylic acid copolymer, a starch derivative such as starch,hydroxyethyl cellulose, sodium alginate, sodium celluloseglycollate, orsodium starchglycollate, a natural polymer such as dextrin, gum Arabic,carrageenan, agar, gelatin, tragacanth or crystalline cellulose, or awater-soluble compound such as glucose, sucrose, fructose, or xylitol.Such material may be coated on an entire or partial surface of the base20, according to the necessity.

By suitably selecting the configuration of the holding layer, it ispossible to attain a controlled release property in the biologicallyactive substance, attached to the holding layer, into a culture liquid.Such controlled release property may also be achieved by adding, at theproviding of the biologically active substance to the sheet, a substancecapable of realizing a controlled release property (for example awater-soluble styrene-acrylic resin) to a liquid containing thebiologically active substance. An amount of the substance for realizingthe controlled release property is 10 mass % or less, preferably 5 mass% or less, to the liquid containing the biologically active substance.

The holding layer can be formed on the sheet 10 by dissolving ordispersing the aforementioned material in a suitable solvent to preparea coating liquid, and coating such coating liquid on the sheet to form acoating film thereon. The coating can be carried out for example by aroll coating, a blade coating, an air knife coating, a gate rollcoating, a bar coating, a size press, a spray coating, a gravurecoating, a curtain coating, a screen printing, a flexographic printingor an offset printing. The thickness of the holding layer can bearbitrarily selected, but is preferably about 1 to 100 μm.

A biologically active substance 22 is depositing on such base 20 and onthe sheet 10. Liquid discharge means can be advantageously employed fordepositing. The liquid discharge means is capable of discharging aliquid droplet of a volume of 100 nl or less per drop, more specifically1 nl or less, such as a micropipette, a microdispenser, or a dischargeapparatus of ink jet method. A discharge apparatus of ink jet method canbe employed particularly advantageously because the discharge apparatusis available inexpensively and a small liquid droplet can be discharged.Furthermore, among the ink jet methods, a thermal ink jet method and apiezo ink jet method can be employed advantageously. A dischargeapparatus of the thermal ink jet method, being easy in preparation offine discharge ports, can discharge a liquid containing a biologicallyactive substance 22 at a high density. Also a discharge apparatus of thepiezo ink jet method, in which a discharge energy is generated by adisplacement of a piezoelectric element, can discharge the biologicallyactive substance without giving a thermal stress thereto.

At the use of the liquid discharge means, the biologically activesubstance 22, 12 is dissolved in an appropriate solvent for discharge.An amount of the dissolved biologically active substance is 10 mass % orless, preferably 5 mass % or less. Any solvent may be employed as longas it can stably dissolve the biologically active substance 22 or 12,but water is employed advantageously. Water is employed in 30 mass % orhigher, preferably 50 mass % or higher. As the water, there arepreferably employed ion-exchanged water (deionized water) or variousbuffers for stably dissolving the biologically active substance 22, 12.

Also the aforementioned water-soluble solvent may be employed ifnecessary.

Also a liquid of the invention containing at least the biologicallyactive substance 22 or 12 may contain at least a hydrophilic resin, inaddition to the aforementioned components. The hydrophilic resin can be,for example, a natural polymer such as a ligninsulfonic acid salt orshellac, a styrene-acrylic acid-acrylate alkyl ester copolymer salt suchas a polyacrylic acid salt, a styrene-acrylic acid copolymer salt, or astyrene-acrylic acid-acrylate ester copolymer salt, an anionic polymersuch as a styrene-maleic acid copolymer salt, a styrene-maleicacid-acrylate alkyl ester copolymer salt, a styrene-maleic acid halfester copolymer salt, a styrene-methacrylic acid copolymer salt, avinylnaphthalene-acrylic acid copolymer salt, a vinylnaphthalene-maleicacid copolymer salt, a β-naphthalenesulfonic acid-formaline condensatesalt, or polyphosphoric acid, polyvinyl alcohol, methylolated melamine,polyvinylpyrrolidone, or a cellulose derivative such as methylcellulose, hydroxymethyl cellulose or carboxymethyl cellulose. In thepresent invention such resins may be employed singly or in a mixture oftwo or more kinds. Also there are many other materials, for example anatural resin such as albumin, gelatin, casein, starch, cationizedstarch, gum Arabic, and sodium alginate. An amount of the hydrophilicresin is 10 mass % or less, preferably 5 mass % or less. Naturally thepresent invention is not limited to such examples.

Such hydrophilic polymer compound can be added also in case of releasingthe biologically active substance 22 from the transfer sheet with acontrolled release property.

Also the liquid of the present embodiment containing at least thebiologically active substance 22 or 12 may further include, if necessaryfor obtaining desired physical properties, a surfactant, a defoamer, anantiseptic, an inorganic salt, an organic salt and the like.

On the cell culture plate, after the biologically active substance 22 isprovided by the liquid discharge means in a desired position on the base20, the biologically active substance 22 is immobilized thereto. Forimmobilizing the biologically active substance 22 to the base 20, atreatment necessary for immobilization may be applied to thebiologically active substance 22 or to the plate. The biologicallyactive substance 22 may be treated to introduce therein a functionalgroup necessary for covalent coupling, such as an amino group, acarboxyl group, a disulfide group, an epoxy group, a carbodiimide group,or a maleimide group, or to bond a chargeable material necessary forcoupling by an electrostatic attractive force, such as fine particles ofa metal or an inorganic oxide, a cationic or anionic polymer. Avidin orbiotin, or an antigen molecule or an antibody molecule can be introducedfor a biological affinity coupling. It is also possible to coat thesubstrate surface with a polymer material or a silane coupling agent andto introduce a functional group necessary for covalent bonding, such asan amino group, a carboxyl group, a disulfide group, an epoxy group, acarbodiimide group, or a maleimide group. It is also possible to form aconductive or semiconductive layer for charging the substrate surface onthe substrate surface, for example, using a metal such as gold, silver,platinum or iron, an inorganic oxide such as indium tin oxide, titaniumoxide or zinc oxide, or a conductive polymer such as polyacetylene,polypyrrole, polyaniline or polythiophene. It is also possible toprovide the surface of the base 20 with a material having a bindingability with the material introduced into the biologically activesubstance 22, such as biotin or avidin, an antibody or antigen, or aprotein having an antibody0binding ability such as protein A.Introduction of such materials provides a firm coupling between thesurface of the base 20 and the biologically active substance 22.

At the immobilization, energy may be applied from the exterior, such aslight radiation or heating. Such energy from the exterior allows toaccelerate the coupling between the surface of the base 20 and thebiologically active substance 22.

The cell culture plate 2 and the transfer sheet 1 can be prepared asdescribed above.

The screening plate 2 and the transfer sheet 1 thus prepared can bestored in a container over a prolonged period. A more preferable stablestorage can be achieved by sealing the transfer sheet in the containertogether with a drying agent or an oxygen removing agent. Also they canbe more simply laminated with a plastic film.

In the following, there will be explained a method of cell culture withthe cell culture kit constituted of the cell culture plate 2 and thetransfer sheet 1 described above. By culturing cells with such kit, thecells are cultured under various influences by the biologically activesubstances. Prior to the cell culture, the transfer sheet and the cellculture plate may be sterilized if necessary for example by radiation orUV irradiation or by washing with alcohol. This operation will preventan inhibition of the culture for example by undesired microorganisms.

Also in the cell culture with the cell culture kit of the cell cultureplate 2 and the transfer sheet 1, the transfer sheet may be immersed inthe culture liquid during the culture, or may be removed in the courseof the culture. Also a desired substance may be added to the cultureliquid of a desired area after the cell culture for a predeterminedperiod. In this manner it is possible to control the concentration ofthe biologically active substance to the cells, to add an activesubstance, to vary the influences or to vary the adhesion to the plate.For adding a desired substance to the culture liquid, there can beemployed a method contacting plural sheets with the culture liquid atdifferent times. It is also possible, in the course of the culture, toreplace the culture liquid and to use a different transfer sheet. Inthis manner, biologically active substances of a different combinationcan be applied at an arbitrary timing of the culture and the cultureconditions can be easily changed. Also in the screening after the cellculture, a desired substance such as an indicator may be added indesired wells. In this manner the screening can be carried out easily.

Also the cultured cells may be removed from the cell culture plateduring or after cell culture. In this manner the removed cultured cellsmay be utilized as an artificially prepared tissue or a part thereof.More specifically, the cultured cells can be removed by treating thecell culture vessel with trypsin after the cell culture. In this manner,the plate can be re-used. Such re-use of the plate is one of theadvantages obtained by immobilizing the biologically active substance tothe plate in such a manner that cells cannot incorporate the activatingsubstance into a metabolic system. Also by coating the plate in advancewith a temperature-responsive polymer such aspoly(N-isopropylacrylamide) and executing the cell culture thereon, itis possible to remove the cultured cells by bringing the temperature toabout 30° C., thereby causing a change in the hydrophilicity of thepolymer surface. In this manner the cells can be utilized for example ina live tissue.

In the following there will be explained a screening method in whichcells are cultured by using the cell culture kit comprised of the cellculture plate 2 and the transfer sheet 1, with a substance immobilizedon the plate and a substance dissolved from the sheet into the cultureliquid. As screening means, there can be utilized the aforementionedmethod of observing the morphological change of the cells cultured onthe cell culture plate 2. For this purpose, there may be employed anymicroscope capable of observing the shape of the cells, not only anoptical microscope but also a scanning electron microscope, atransmission electron microscope, a scanning probe microscope or afluorescence microscope. A screening plate bearing cultured cells isplaced in an observing position of such microscope and the shape of thecells is observed under the microscope. Microscopic observation of thecell morphology alone is sufficient for screening, enabling simpleevaluation. The cells may be stained for evaluation. Cell stainingfacilitates evaluation under the microscope in case the cellsproliferates in a high density or in case the cells causes a fusion bydifferentiation thereby forming a polykaryocyte.

Alternatively, there may be utilized a quantitative determination of asubstance produced by the cells or incorporated therein in the course ofor as a result of cell adhesion to the culture plate or proliferation ordifferentiation. In case an object of quantitative determination cannotbe directly evaluated, the quantitative determination may be carried outwith a reporter substance. As a specific example, a desired protein canbe quantitatively measured by introducing a gene of a measurable proteinin the vicinity of the gene of the desired protein to be measured by thegenetic engineering technology, and by quantitatively determining theexpressed measurable protein. Evaluation of such substances allows adetailed survey of changes in the cells induced by the substanceimmobilized on the base, and leads to elucidation of the informationtransmission mechanism in the cells. In case of carrying out evaluationwith a substance incorporated into the cells, it is also possible tolabel the substance with a labeling material enabling evaluation,whereby the quantitative determination becomes rather easy.

For a quantitative determination of these substances, there may beemployed a method of measuring an amount of a radiation emitted from aradioactive compound, a method of measuring an amount of fluorescenceemitted from a substance labeled with a fluorescent substance, a methodof measuring an amount of light emitted from a light-emitting substance,or a method of measuring an optical absorbance of a dye.

A method that employs a radioactive compound containing a radioactiveisotope of an element abundantly present in a live tissue such ashydrogen, carbon, nitrogen, phosphor or sulfur and measures theintensity of radiation from such a compound is highly sensitive, andallows observation of phenomena occurring in a live body, because such ahot compound has the same chemical properties as the cold compound.

Also a method of labeling with a fluorescent substance is relativelysimple and gives little influence on the metabolism of the cells byemploying a fluorescent substance of a low molecular weight. Also in aquantitative determination of a substance produced by the cells by adetermination method utilizing an antigen-antibody reaction, anevaluation by a fluorescent measurement is effective since antibodieslabeled with a fluorescent substance are available in various kinds andprovide a high measuring sensitivity.

Also the method of measuring luminescence from a luminescent substanceallows to recognize even a small change, since the luminescence can bemeasured with a high sensitivity. When a gene expressed with celladhesion, proliferation, differentiation or substance production causedby a substance has been specified, it is possible to introduce a firebugluciferase gene or the like in the vicinity of such a gene and an amountof luciferase produced by the gene expression is measured from theintensity of luminescence generated on addition of ATP and luciferin. Inthis manner it is possible to evaluate the influence of the screenedsubstances from the luminescence intensity.

In a method of measuring the optical absorbance of a dye, it is possibleto amplify the optical absorbance of a dye for example by employing anenzyme reaction in combination, thereby enabling a quantitativedetermination of a substance of a very small amount.

EXAMPLES

In the following, the present invention will be clarified further byexamples thereof, but such examples are merely given for the purpose ofdeeper understanding of the invention and the present invention is notlimited to such examples.

Example 1

The following method was employed for mixing one or more biologicallyactive substances in combination into a cell culture liquid. Basicfibroblast cell growth factor (b-FGF), insulin-like growth factor(IGF-I) and bone morphogenetic protein (BMP-2) were used as thebiologically active substance. b-FGF was dissolved in a physiologicalsaline solution, IGF-I in 10 mM acetic acid, and BMP-2 in 4 mMhydrochloric acid to prepare solutions each containing the biologicallyactive substance at a concentration of 20 μg/ml and 5% glycerin. Inkcartridges were washed with 70% ethanol, to which the respectivesolutions prepared above were filled.

Then, on a base of a polyolefin sheet (NOVIX-II® manufactured by AsahiTechno Glass), sterilized in advance with a sterilizing lamp, thebiologically active substances were discharged by using an ink jetprinter (Ink jet printer F930, Canon). The amount of the biologicallyactive substance was controlled by the printing area and the number ofdischarge, and 27 areas different in kinds and concentrations of thebiologically active substances were formed on the transfer sheet bysuperimposed discharge.

A DMEM (Dulbecco's modified Eagle's minimum essential medium) culturemedium containing 2% FBS (fetal bovine serum) was put in wells of a96-well transparent microplate (SUMILON for cell culture).

The transfer sheet was tightly placed on the microplate in such a mannerthat the 27 areas corresponded to the wells of the microplate, and theywere lightly shaken to make the culture liquid contact with the transfersheet and dissolve the biologically active substances into the cultureliquid. Then, in each well, cells of mouse skeletal muscle cell strainC2C12 were cultured for 96 hours at 37° C., in humidified air suppliedwith 5% CO₂.

Microscope observation of the microplate after the culture showed cellproliferation in the areas containing b-FGF, differentiation into musclecells in the areas containing IGF-I, and differentiation into bone cellsin the area containing BMP-2, respectively accelerated depending on theconcentrations.

The cells after the culture were treated with 10% formalin for 15minutes and then with methanol for 15 minutes, and finally with a1000-fold dilution of fluorescent dye (TOTO-3 manufactured by MolecularProbe) for 30 minutes for fluorescent staining of DNA. The fluorescentintensity of TOTO-3 at 700 nm was employed as an index for the cellproliferation. Also the cells were subjected to freeze-fracture and thecreatinkinase (KP) and alkaliphosphatase (ALP) activities were measured.Relative activity thereof based on the total protein content in thesolution was taken as an index of differentiation, i.e., the KP activityfor muscle differentiation and the ALP activity for bonedifferentiation.

As a result, it was shown that, in areas where the three factors werepresent in various concentrations, the cell differentiation proceeded ina different direction compared with areas where only one factor waspresent. Use of such a transfer sheet allows to investigate theinteraction of plural biologically active substances at the same time.

Example 2

The following process was employed for investigating the influences ofbiologically active substances on cells when the species andconcentrations of the active substances were varied in a predeterminedperiod.

As the biologically active substances, fibroblast cell growth factor-2(FGF-2), insulin-like growth factor-I (IGF-I), bone morphogeneticprotein-2 (BMP-2) and transforming growth factor-β (TGF-β) wereemployed. Solutions of these biologically active substances wereprepared and filled in ink cartridges as in Example 1.

Then, two polyolefin sheets (NOVIX-II® manufactured by Asahi TechnoGlass) were coated with polyvinyl alcohol (Gosenol GM14L, manufacturedby Nippon Gosei Kagaku) by using a bar coater to a dry film thickness of5 μm, thereby preparing two sheet bases having a holding layer. Then,the sheets were sterilized with a sterilizing lamp and the biologicallyactive substances were discharged onto them using an ink jet printer.Thus two transfer sheets were prepared, a transfer sheet A having FGF-2and IGF-I in various combinations and concentrations in 12 areas, and atransfer sheet B having BMP-2 and TGF-β in various combinations andconcentrations in 12 areas.

A DMEM culture liquid containing 2% FBS was put into wells of two96-well transparent microplates 1 and 2. After the transfer sheets A andB were attached onto the microplates 1 and 2 respectively as in Example1, the biologically active substances were dissolved by pushing eacharea carrying the biologically active substances from the back of thesheet with a rod (FIG. 5). In this manner, the sheet A was used toprovide FGF-2 and IGF-I to the microplate 1, and the sheet B was used toprovide BMP-2 and TGF-β to the microplate 2. Then, in each well of eachmicroplate, cells of mouse skeletal muscle cell strain C2C12 werecultured for 48 hours in humidified air supplied with 5% CO₂. 48 hoursafter the start of the culture, the culture liquid was removed from eachcell by suction. After fresh 2% FBS/DMEM was added to each well, atransfer sheet B was applied to the microplate 1 to supply BMP-2 andTGF-β, while a transfer sheet B was applied to the microplate 2 tosupply FGF-2 and IGF-I, and the cell culture was conducted for further48 hours. After the culture, cells were frozen and disrupted and CK andALP activities thereof were measured to use relative activities asindexes for muscle differentiation and bone differentiationrespectively.

As a result, muscle differentiation was accelerated in areas containingIGF-I only, and bone differentiation was accelerated in areas containingBMP-2 only. It was confirmed that, when four factors acted on the cellsin various combinations and concentrations in a time sequential manner,the cell differentiation proceeded differently in comparison with caseswhere these factors acted at the same time.

Use of such transfer sheets allows to change the kinds and theconcentrations of the substances acting under plural conditions within apredetermined time.

Example 3

The following process was employed for investigating the influences ofbiologically active substances on cells when the species andconcentrations of the active substances were varied in a predeterminedperiod and active substances were gradually released.

As the biologically active substances, there were employed bonemorphogenetic protein (BMP-2), fibroblast cell growth factor (FGF-2),and insulin-like growth factor (IGF-I). The BMP-2 and FGF-2 wererespectively prepared into 20 μg/ml solutions containing 5% glycerin. Onthe other hand, the 20 μg/ml solution of the insulin-like growth factor(IGF-I) contained 5% glycerin and 3% styrene-acrylic resinmonoethanolamine salt (average molecular weight 8,000, acid value 250)for controlled release of IGF. Ink cartridges were washed with 70%ethanol, and were filled with the respective solutions of thebiologically active substances.

Then, a plurality of transfer sheets each containing a singlebiologically active substance in four different concentrations wereprepared by providing on a base of a polyolefin sheet the respectivebiologically active substance using an ink jet printer as in Example 1.Each polyolefin sheet was processed in advance to have minute holes atpositions corresponding to the wells of a 96 well plate to allow gasexchange. A DMEM culture liquid containing FBS by 2% was put in wells offive 96-well transparent microplates. Then the transfer sheet carryingBMP-2 was placed on each of the 96-well transparent microplates tosuperimpose on the wells of the microplate. The sheet was pressed fromthe above using a jig having 96 protrusions at positions correspondingto the wells of the microplate, so that each region of the sheet came incontact with the culture liquid in each well to dissolve BMP-2 into theculture liquid. Then, in each well, cells of a mouse skeletal musclecell strain C2C12 were added. Next, a transfer sheet carrying IGF-I wassuperimposed on each of the microplate and pressed from the above usingthe above jig so that each region of the sheet came in contact with theculture liquid in each well to gradually release IGF-I into the cultureliquid, and cell culture was started. The above operations were carriedout in the same manner with the five microplates. After a predeterminedtime from the start of the culture, the transfer sheet of one of themicroplates was replaced by a transfer sheet carrying. FGF-2 to dissolveFGF-2 into the culture liquid. After that the transfer sheet wasreplaced by the previous IGF-I sheet and the culture was continued. Allthe cells were cultured for 114 hours in total at 37° C., in humidifiedair containing 5% CO₂. The FGF-2 addition was conducted at the followingpoints:

-   -   (1) immediately after the start of culture;    -   (2) 12 hours after the start of culture;    -   (3) 24 hours after the start of culture;    -   (4) 48 hours after the start of culture;    -   (5) 72 hours after the start of culture.

The cells after the culture were treated with 10% formalin for 10minutes and staining with the enzyme activity of alkaliphosphatase (ALP)was carried out as an index for bone differentiation by BMP-2.

As a result, the influence on the bone differentiation changed by thetiming of action of FGF-2 under sustained release of the insulin-likegrowth factor-I (IGF-I). In the case without addition of FGF-2 and inthe aforementioned conditions (3), (4) and (5), portions stained withALP were confirmed, but the stained portions were scarcely observed in(1) and (2). These results indicate that, at the differentiation fromthe muscle cells into bone cells, the bone differentiation issignificantly suppressed by adding FGF-2 within 24 hours after the startof the culture. It is thus possible, by transfer sheets capable ofadding the biologically active substance at different times, toinvestigate the effect of the action period and the effect of controlledrelease.

Example 4

In this example, potential allergens provided on transfer sheets weretransferred to a culture liquid, in which cells were cultured toquantitatively determine histamine, an inflammation-inducing substanceproduced by sensitive cells, thereby evaluating whether a subject has anallergy. This example evaluated allergy to cedar pollen, cow milk, housedust and ambrosia. The cedar pollen, house dust and ambrosia weresufficiently ground in advance with a homogenizer. An aqueous solutioncontaining each allergen was centrifuged, and a soluble component wasobtained by eliminating a precipitate. Then the solution of each solubleallergen component was diluted with 50% methanol into a concentration of50 μg/ml, and a transfer sheet was prepared by forming 4 areas ofrespective allergen components and 6 areas of two allergen combinationsof the four allergens on a 20 μm thick polyester film using an ink jetprinter. The blood sample of an object person were subjected to adensity gradient centrifugation to separate blood components, therebyobtaining allergy-reactive cells.

A DMEM culture liquid containing FBS (fetal bovine serum) by 10% was putinto wells of a 12-well transparent microplate. The transfer sheetprepared above was placed on the 12-well transparent microplate suchthat areas were superimposed onto the wells of the microplate. The sheetand plate was lightly shaken to contact the culture liquid with thetransfer sheet, thereby dissolving the biologically active substances inthe culture liquid. Then, in each cell, the allergen reactive cells werecultured for 96 hours at 37° C., in humidified air containing 5% CO₂.The plate after culture was taken out and rinsed with an isotonicphosphate buffer. After a treatment with methanol for 30 minutes anddrying, the cells were treated with a rabbit anti-histamine antibody for1 hour, and washed with PBS followed by a treatment with an anti-rabbitIg antibody labeled with horse radish peroxydase for 1 hour. Then ahistamine amount was determined from an absorbance change ofo-phenylenediamine using an enzyme-antibody process. As a result,histamine was found in a large amount in the area containing house dustalone, so that the person was considered allergic to the house dust.Also the result was negative for the cedar pollen, cow milk or ambrosiaalone, but histamine was detected in an area where ambrosia and cow milkwere combined, so that a possibility of allergy was identified in caseof taking these two at the same time. In this manner, the transfer sheetcan be used to diagnose the cause of allergy in a simple manner. Inparticular, as shown in this example, allergy reaction to a combinationof plural allergens can be easily investigated.

Example 5

The following method was employed for acting biologically activesubstances in combination on cells. As the biologically activesubstances, there were employed basic fibroblast cell growth factor(b-FGF), insulin-like growth factor (IGF-I) and bone morphogeneticprotein (BMP-2).

First, bFGF and IGF-I were immobilized onto a polystyrene 96-well cellculture plate in the following manner.

On the cell culture plate coated with poly-L-lysine, a solution (1.5mg/ml) of active dextran activated with tresyl chloride was added tobind the activated dextran to the plate. Then carbonate buffer solutionscontaining b-FGF and IGF-I respectively were prepared, applied to themicroplate with various amounts. The microplate was left to stand for 12hours at 4° C. to prepare a cell culture plate including areas whereb-FGF and IGF-I were immobilized either singly or in combination withdifferent concentrations, as well as control areas withoutimmobilization.

A polystyrene sheet having projections corresponding the wells of themicroplate was provided and sterilized. Then a solution containing 5%glycerin and 20 μg/ml BMP-2 was prepared and filled in an ink cartridgewashed with 70% ethanol. Then BMP-2 was provided by using a Canon inkjet printer PIXUS950i onto the projections of the polystyrene sheet,thereby obtaining a transfer sheet.

A culture liquid DMEM containing FBS by 2% was put in the wells of the96-well cell culture plate prepared above.

The transfer sheet was placed on the 96-well transparent microplate insuch a manner that each projection made a pair with a well of themicroplate and contacted with the wall of the well. Thus the area of thebiologically active substance formed on each projection was brought intocontact with the culture liquid, and BMP-2 was dissolved in the cultureliquid with shaking for accelerating dissolution of the biologicallyactive substance. Then cells of mouse skeletal muscle cell strain C2C12suspended in DMEM (Delbucco's modified eagle's minimum essential medium)supplemented with 2% FBS (fetal bovine serum) were added to each well to500 cells/well, and was cultured for 96 hours at 37° C., in humidifiedair containing 5% CO₂.

After the culture, the cell culture plate was observed under an opticalmicroscope to show cell proliferation in areas containing b-FGF, muscledifferentiation in areas containing IGF-I, and bone differentiation inareas containing BMP-2 depending on the concentration thereof. Then thecells were treated with 10% formalin for 15 minutes and then withmethanol for 15 minutes, and reacted with a fluorescent dye for 30minutes for fluorescent staining of DNA. Fluorescent intensity of DNA at700 nm was measured as an index for cell proliferation. Also the cellswere subjected to freeze-fracture to measure the creatinkinase (KP) andalkaliphosphatase (ALP) activities. Relative activity thereof based onthe total protein content in the solution was taken as an index ofdifferentiation, i.e., the KP activity for muscle differentiation andthe ALP activity for bone differentiation. Thus the complex effects ofthree factors, namely immobilized bFGF, soluble IGF-I and BMP-2, to thecells were analyzed.

Example 6

An epidermal cell growth factor (EGF) and a nerve cell growth factor(NGF) were employed as the biologically active substances. A cultureplate of polystyrene 96-well cell culture plate was prepared by aprocess similar to that in Example 5, providing areas (wells) where EGFwas immobilized with stepwise concentrations, and control areas (wells)of no immobilization. Separately, a stretchable polyolefin sheet wascoated with hydroxypropyl cellulose by using a bar coater and heated for10 minutes at 60° C. to form a holding layer. Then EGF and NGF wereprovided on the sheet by using a Canon ink jet printer PIXUS950i toobtain a transfer sheet on which areas carrying EGF and NGF singly or incombination. Screening was conducted with these cell culture plate andtransfer sheet.

The transfer sheet was placed on the cell culture plate sterilized inadvance with a sterilizing lamp and filled with a culture liquid, thenthe areas carrying biological substance(s) of the sheet were broughtinto contact with the culture liquid in the wells by pushing theopposite surface of the sheet with a pointed article, and the contactwas maintained for 15 minutes under light shaking to dissolve thebiologically active substance in the culture liquid. Then cells of anerve cell strain PC12 suspended in RPMI (Rosewell Park MemorialInstitute) 1640 culture medium containing 2% FBS were added to each wellto 500 cells/well, and cultured for 48 hours at 37° C., in humidifiedair containing 5% CO₂. Then the culture liquid was removed, and thecells were immobilized and stained with hematoxilin-eosin for evaluatingthe level of proliferation and differentiation, and the plate wasobserved under an optical microscope on the nuclei and cell morphology.As a result, it was confirmed that the EGF in an immobilized stateaffected the PC12 cells differently from that in a dissolved state. Inthis manner, effects of a growth factor in a dissolved state and animmobilized state on cells could be investigated in a simple manner.

Example 7

A rat was immunized with mouse bFGF as an antigen, and after two monthsthe spleen cells were fused with myeloma cells to obtain hybridomas,from which a large number of monoclonal antibody producing hybridomaswere obtained and 96 monoclonal antibodies were purified. Then, mousebFGF was immobilized at: 200 pg/mm² on a polystyrene 96-well plate toprepare a culture plate, Separately, a para film was coated withhydroxypropyl cellulose on the entire surface by using a bar coater, onwhich the obtained monoclonal antibodies were provided by using a Canonink jet printer PIXUS950i at positions corresponding to the wells of the96-well plate. 75 μl of DMEM culture medium was added to each well ofthe culture plate, and the sheet was placed on the culture plate, andthe culture liquid in the well was brought into contact with theantibody area of the screening sheet, and the antibody was transferredinto the culture liquid.

Then 75 μl of DMEM medium containing muscle cells of strain C2C12 at6×10⁴ cell/ml was added to each well, and cell culture was conducted at37° C. in humidified air containing 5% CO₂. After 48 hours, 0.002%5′-bromo-2′-deoxyuridine (BrdU) was added to the culture liquid, after 3hours the culture liquid was removed and the cells were treated withmethanol for 30 minutes. Staining of the cells with an FITC labeledanti-BrdU antibody was conducted for evaluating cell proliferation, andthe nuclei were stained with a 10,000-fold dilution of Hoechst 33258 for5 minutes. The excessive staining solution was washed off with anisotonic phosphate buffer. Thus prepared plate was observed under afluorescent microscope to evaluate the number of the stained nuclei.Also the number of nuclei containing BrdU-labeled DNA was determined byfluorescent analysis.

As a result, in three wells the fluorescence of BrdU was low incomparison with other wells showing that monoclonal antibodies A, B andC corresponding to these wells were neutralizing antibodies thatefficiently inhibited the growth promoting effect of bFGF.

Example 8

In order to investigate the concentration dependence of thebFGF-inhibiting effect of the three neutralization antibodies obtainedin Example 7, there was prepared a cell culture plate in which bFGF wasimmobilized at three different concentrations in the wells of apolystyrene 96-well plate. Separately, a parafilm was coated withhydroxypropyl cellulose on the entire surface by using a bar coater, onwhich the obtained monoclonal antibodies A, B and C were provided byusing a Canon ink jet printer PIXUS950i at positions corresponding tothe wells of the 96-well plate. 75 μl of DMEM culture medium was addedto each well of the culture plate, and the sheet was placed on theculture plate, and the culture liquid in the well was brought intocontact with the antibody area of the screening sheet, and each antibodywas transferred into the culture liquid.

Then 75 μl of DMEM medium containing muscle cells of strain C2C12 at6×10⁴ cell/ml was added to each well, and cell culture was conducted at37° C. in humidified air containing 5% CO₂.

After 48 hours, the culture liquid was removed, and the cells weretreated with methanol for 30 minutes, dried and immobilized. Forevaluating cell proliferation, the nuclei were stained with a10,000-fold dilution of Hoechst 33258 for 5 minutes. The excessivestaining solution was washed off with an isotonic phosphate buffer. Thusprepared plate was observed under a fluorescent microscope to evaluatenumber of the stained nuclei.

As a result, in the wells containing the transferred bFGF neutralizingantibody, the number of cells decreased with an increase in theconcentration of each neutralizing antibody, indicating inhibition ofthe cell proliferation-accelerating effect of bFGF. Also the comparisonof the cell numbers indicated that the strength of inhibition was in anorder of B>C>A.

The present invention is not limited to the above embodiments andvarious changes and modifications can be made within the spirit andscope of the present invention. Therefore, to appraise the scope of thepresent invention, the following claims are made.

This application claims priority from Japanese Patent Applications No.2003-418523 filed Dec. 16, 2003 and No. 2003-418535 filed on Dec. 16,2003, which are hereby incorporated by reference herein.

1. An elastic transfer sheet that transfers a biologically activesubstance that affects a function of a cell to a culture region on aculture plate when placed on the culture plate, the elastic transfersheet comprising: a sheet base; and two or more holding areas providedon the sheet base, each of the two or more holding areas holding abiologically active substance, wherein each of the two or more holdingareas is provided in a position for covering a culture region of theculture plate, wherein the biologically active substance is releasablefrom the elastic transfer sheet, and wherein each of the two or moreholding areas is surrounded by its own protruding wall structure.
 2. Thetransfer sheet according to claim 1, wherein the two or more holdingareas hold different biologically active substances or differentcombinations of two or more biologically active substances.
 3. Thetransfer sheet according to claim 1, wherein the two or more holdingareas hold a biologically active substance in different concentrations.4. The transfer sheet according to claim 1, wherein each of the two ormore holding areas is able to release the biologically active substancein a sustainable manner.
 5. The transfer sheet according to claim 1,wherein each of the two or more holding areas is a protruding areaprovided on the sheet base, and the biologically active substance isheld on the protruding area.
 6. A method for applying a biologicallyactive substance to the transfer sheet according to claim 1, the methodcomprising a step of providing each of the two or more holding areas ofthe transfer sheet with the biologically active substance by usingliquid discharge means.
 7. The method according to claim 6, wherein theliquid discharge means is discharge means by a thermal ink jet means. 8.The method according to claim 6, wherein the liquid discharge means isdischarge means by a piezo ink jet means.
 9. The method according toclaim 6, further comprising a step of immobilizing the biologicallyactive substance by applying an immobilizing energy from the exterior.